Mycobacteria are gram-positive, acid-fast rods that do not produce flagella, capsules or spores and do not grow on ordinary microbiological media. They are strict aerobes with nutritionally-exacting growth requirements, typically requiring an enriched medium for culturing. Mycobacteria comprise both slow-growing and fast-growing species. Fast-growing organisms, forming colonies in <7 days, are generally regarded as non-pathogenic. The slow-growing organisms, however, are those of clinical interest, as they are causal agents of some chronic infections, including human tuberculosis (M. tuberculosis), non-human tuberculosis (M. bovis), leprosy (M leprae) and Johne's Disease (Mycobacterium avium subsp. paratuberculosis; MAP).
Because of the slow growth rate and the need for an enriched medium for mycobacterial growth in culture, detection of Mycobacteria from clinical samples (e.g. sputum, lung fluids, tissue or feces) represents a significant biological challenge. The challenge arises from the fact that more rapidly-growing bacteria can overgrow the slow-growing mycobacterial organism of interest, thus precluding or significantly hindering Mycobacteria detection. Over the decades, several techniques have been developed to decontaminate diagnostic samples (i.e. kill or inhibit non-mycobacterial organisms) submitted for mycobacterial identification. These techniques either kill the potential contaminates or injure them to the extent that their growth is inhibited or totally prevented. For M. tuberculosis, for instance, one decontamination process involves processing the sample in 4% sodium hydroxide for several hours, then neutralizing the sodium hydroxide with oxalic acid. After neutralization, a potent cocktail of antibiotics referred to as PANTA (polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin) is added to the sample to further reduce the growth of contaminating bacteria.
For MAP, one decontamination method involves a three-day protocol that includes processing samples with 0.9% hexadecylpyridinium chloride (HPC; a detergent), centrifuging them and then a double incubation with BHI (brain heart infusion) and antibiotic brew (ANV; amphotericin B, nalidixic acid and vancomycin). Unfortunately, while the MAP decontamination procedures are effective, MAP colony forming unit (cfu) counts are reduced as a result of the decontamination procedure. Most experts agree that a reduction of 1–2 logs of MAP is a typical result of such processing. This reduction potentially lengthens the duration of culturing necessary to detect mycobacterial growth, or in the worse case, renders the Mycobacteria non-detectable. Neither outcome is desirable.
Olstein et al. (U.S. patent publication 2003/0175207) disclose the use of bacteriocin-metal complexes for use in detecting bacteria, including mycobacteria, in samples. Imboden et al. (U.S. patent publication 2005/0014932) disclose the use of fusion proteins comprising a bacteriocin as biocides to kill pathogens, including mycobacteria. Neither reference discloses methods of mycobacterial cultures with reduced contamination.
In light of these findings, it is clear that, prior to the present invention, there was an unmet need in the art for a method of culturing Mycobacteria that reduces non-mycobacterial contamination, without significantly effect on the Mycobacteria. The instant invention meets this need.